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Ann. Rev. MiC'robiol 1982. 36:101-23

Copyright 1982 by Annual Reviews Inc. All rights reserved

PRIMARY AMEBIC
MENINGOENCEPHALITIS AND THE
BIOLOGY OF NAEGLERIA FOWLERI
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David 1: John
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Department of Microbiology, Medical College of Virginia, Virginia

Commonwealth University, Richmond, Virginia 23298

CONTENTS

INTRODUCTION ......................................................................................................... . 101

HUMAN INFECTION ................................................................................................. . 102
Clinical Features ......... .. ....... .................. . ....... . ........... . ........ . ..................................... . 103
103
fJ:!:! ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::.
Treatme.t .................................................................................................................
104
105
EPIDEMIOLOGY AND ECOLOGY ......................................................................... . 106
Geographic Distribution .. .. . .. . . .... ............. . ... .. ..... .......... .. ......... . .. ...... ....... . ..... .. .. .. ....... . 106
Environmental Isolations ........................................................................................... . 106
Animal Infection ........................................................................................................ 107
Control and Prevention ............................................................................................ .. 108
PHYSIOLOGY/METABOLISM ................................................................................. . 109
Nutrition and Growth ............................................................................................... . 109
Cell Difj"erentiation .... .. ......... ................... . ................................................... .. . .... ..... .. 111
Macromolecular Composition .. . . . . . .. . ................................................................. .. ..... .. 113
Cell Membrane and Agglutination ............................................................................ 113
Respiratory Metabolism ........ ............... . .............. . .......................................... ..... ....... . 113
VIRULENCE AND IMMUNITY ............................................................................... . 114
Mechanisms of Pathogenesis ..................................................................................... . 114
Resistance and Susceptibility............... . . ... . ... .......... . .... . ........... . ....... . ...................... . . . . 117
Immunization and Protection .................................................................................... 118
CONCLUDING REMARKS ....................................................................................... . 119

INTRODUCTION

Primary amebic meningoencephalitis (PAM) is a rapidly fatal human dis

ease caus(:d by the ameboflagellate Naegleria fowleri. The disease first was
detected in man 17 years ago by Fowler & Carter in Australia (56). A year
later in 1966 three fatal infections were described from Florida by Butt (10).
101
0066-4227/82/1001-0101$02.00
 102 JOHN

The symptomatology of these cases was remarkably similar to that observed

in Australia. Although it was not apparent then, the seven cases in Australia
and Florida provided almost a complete array of the important clinical and
pathological features of the disease.
Notable, also, was the indication that infection was acquired by intrana
sal instillation during swimming. Butt (10) recognized the discovery of a
new disease in Australia and Florida by contributing a new name, primary
amebic meningoencephalitis.
Naegleria fowleri was named in honor of Dr. Malcom Fowler who first
recognized the disease it caused (13) . This has been the only species of
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

Naegleria to be isolated from victims of PAM. Synonyms for N fowleri are

N aerobia (114) and N. invades (24) . Nonpathogenic species of Naegleria
include N. gruberi (see 58) , possibly the most common ameba in fresh water
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(101) , N. thorn toni (113) , and N. lovaniensis (118) . N. jadini. isolated from
swimming pool water in Belgium, is only slightly pathogenic for mice (140) .
Naegleria is an ameboflagellate; it belongs to a family of amebae (VaW
kamprudae) whose members can transform from amebae into flagellates
(l01) . The Naegleria flagellate is a transient, nonfeeding, nondividing form.
The life cycle of Naegleria also includes cyst formation and excystment by
amebae (Figure 1) . Naegleria flagellates do not encyst; only the amebae
divide and are able to encyst.

HUMAN INFECTION
Amebic infections of the central nervous system may be caused by the
parasitic ameba Entamoeba histolytica or by the opportunistic free-living
amebae Naegleriafowleri or Acanthamoeba spp. E. histolytica may produce
a brain abscess after extraintestinal invasion and subsequent hematogenous

FLAGELLATE

Figure 1 Life cycle of Naeg/eria low/eri.

 NAEGLERIA MENINGOENCEPHALITIS 103

spread. Acanthamoeba spp. produce an illness known as granulomatous

amebic encephalitis (9 1) . Disease usually occurs in chronically ill or debili
tated individuals, some of whom may be undergoing immunosuppressive
therapy. Invasion of the central nervous system appears to be hematoge
nous, arising from a primary lesion of the skin, lungs, or kidneys. In this
review, discussion is limited to N. fowleri and the disease it produces.

Clinical Features
PAM typically occurs in healthy children or young adults with a recent
history of swimming in freshwater lakes or pools. The disease is rapidly
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fatal, usually producing death within 72 hr after the onset of symptoms.

Infection follows inhalation of water containing amebae or flagellates. It
also has been suggested that inhaling cysts, during dusts storms, for exam
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ple, could lead to infection (S 4, lOS).

Amebal penetrate the nasal mucosa and the cribiform plate and travel
along the olfactory nerves to the brain. Amebae first invade the olfactory
bulbs and then spread to the more posterior regions of the brain. Within
the brain they provoke inflammation and cause extensive damage to the
tissue (13.,
The clinical course is dramatic. Symptoms begin with severe frontal
headache, fever (39 -40C) , and anorexia. This is followed by nausea, vomit
ing, and signs of meningeal irritation. Involvement of the olfactory lobes
may caust: disturbances in the sense of smell or taste and may be noted early
in the course of the disease. Visual disturbances may occur. The patient may
experiencl confusion, irritability, and restlessness and may become irratio
nal before: lapsing into coma. Generalized seizures also may be present. In
order of frequency of occurrence, the more important symptoms include
headache, anorexia, nausea, vomiting, fever, and stiff neck (49 , 9 3) .

Pathology
The gross pathologic findings in PAM are remarkably constant. The cere
bral hemispheres usually are edematous and swollen. Meninges are diffusely
hyperemic with a slight purulent exudate. The cortex contains many focal
superficial hemorrhages. The olfactory bulbs exhibit marked involvement
with hemorrhage, necrosis, and purulent exudate (14, 49 , 9 3).
Micros1cope examination reveals many amebae in the subarachnoid and
perivascular spaces. Presumably, the perivascular spaces provide a path of
migration for the amebae, and the blood vessels supply the oxygen needed
by these Lerobic organisms. In fewer numbers, amebae are found clustered
within the brain tissue and in the purulent exudate of the meninges and
brain substance. Within the exudate some amebae may be seen engulfed by
macrophages. Many amebae are observed to contain phagocytosed cellular
 104 JOHN

debris and erythrocytes. The purulent exudate contains numerous polymor

phonuclear and mononuclear leukocytes (14, 9 3) .
The cortical gray matter is a preferred site for amebae development;
consequently, severe involvement occurs in the cerebral hemispheres, cere
bellum, brain stem, and upper portions of the spinal cord. Encephalitis
ranges from slight amebic invasion and inflammation to massive invasion
with purulent, hemorrhagic necrosis. Typically, the olfactory bulbs exhibit
extensive amebic invasion, hemorrhage, and an inflammatory exudate; the
involvement here is greater than in other areas of the brain. Infection of the
central nervous system with N. fowleri may be described best as an acute,
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

hemorrhagic, necrotizing meningoencephalitis (14, 9 3) .

Focal demyelination in the white matter of the brain and spinal cord has
been described (27,51). Curiously, demyelination occurred in the absence
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of amebae or cellular infiltration. Chang (27) suggests that demyelination

is caused by a phospholipolytic enzyme or enzyme-like substance produced
by actively growing amebae present in the adjacent gray matter.
Myocarditis has been described in some patients dying of PAM (14,89 ) .
It has been suggested (89) that the observed myocarditis may be caused by
a circulating myotoxin either produced by amebae in the brain or released
by rapidly degenerating invading amebae. However, evidence has not yet
been produced to substantiate either hypothesis.

Diagnosis
The diagnosis of PAM is made by microscope identification of living or
stained amebae in patient cerebrospinal fluid. Motile amebae are readily
seen in simple wet-mount preparations of spinal fluid. Amebae can be
distinguished from other cells by their limax (L. sluglike) shape and pro
gressive movement. It is not necessary to warm the slide since amebae
remain fully active at room temperature. Refri gerati on of the spinal fluid
is not recommended as this may kill the amebae (13, 9 3) .
Spinal fluid smears may be stained with Wright or Giemsa stains (108) .
The bacterial Gram stain is of little positive value since heat fixing causes
the amebae to stain poorly and to appear as degenerate cells. Giemsa- or
Wright-stained amebae have considerable amounts of sky-blue cytoplasm
and relatively small, delicate, pink nuclei. Mononuclear leukocytes, on the
other hand, have large purplish nuclei with only small amounts of sky-blue
cytoplasm. Acridine orange has also been used to distinguish N. fowleri
amebae from leukocytes (9 7) . Using acridine orange and ultraviolet micros
copy, amebae stain brick-red with pale green nuclei in contrast to leuko
cytes, which are bright green.
Amebae also may be cultured by placing some of the spinal fluid on
non-nutrient agar (1.5%) spread with a lawn of Enterobacter aerogenes or
 NAEGLERIA MENINGOENCEPHALITIS 105

Escherich.ia coli and incubated at 37C. The amebae will grow on the moist
agar surfa.ce and will utilize the bacteria as a source of food.
Clinically, PAM very closely resembles fulminating bacterial meningitis,
and the laboratory findings are also similar. The cerebrospinal fluid is
purulent or sanguinopurulent with leukocyte counts, predominantly neu
trophils, ranging from a few hundred to over 20,000 cells/mm3. Spinal fluid
glucose levels are low, and generally, protein content is increased. Typically,
Gram-stained smears and cultures of spinal fluid are negative for bacteria
(14, 93).
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

Treatment
At present, no satisfactory treatment for PAM exists. The antibiotics used
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to treat ba.cterial meningitis are ineffective in naeglerial infection, as are the

antiamebk drugs. Amphotericin B, a drug of considerable toxicity, is an
antinaegle:rial agent for which there is some evidence of clinical effective
ness. Both known survivors of PAM were treated with amphotericin B,
given intravenously and intrathecally (2, 15); the only patient in the United
States known to have survived naeglerial infection (15) also was given
parenteral miconazole and oral rifampin. The in vitro testing of a highly
virulent human isolate of N. fowleri demonstrated that amebae were ex
tremely susceptible to amphotericin B [minimal inhibitory concentration
(MIC), 0.15 /Lglml] , somewhat susceptible to miconazole (MIC, 25
/Lglml), aLnd resistant to rifampin (MIC, <100 /L/ml) (119). Mice were
protected by treatment with amphotericin (7.5 mglkg/day) but not by
treatment with lower doses of amphotericin B alone or in combination with
miconazole (100 mglkg) or rifampin (220 mglkg). It appears, then, that
amphotericin B currently is the most effective treatment for PAM. Am
photericin B is administered intravenously at a dose of 1 mg per kg of body
weight daily, with intrathecal inoculations of 0.1 mg on alternate days (14).
Amphotericin B is a polyene compound that acts upon the plasma mem
brane, disrupting its selective permeability, and causing leakage of cellular
components (82). When exposed to amphotericin B, amebae round up and
do not form pseudopodia. Membrane-related changes, evident by electron
microscopy, include enhanced nuclear plasticity, increased amount of
smooth and rough endoplasmic reticulum, decreased food vacuole forma
tion, and production of blebs of the plasma membrane (110).
Lipopolysaccharide has been shown to afford mice some protection for
several days after challenge with N. fowleri (1). Slightly better protection
was provided by treatment of mice with 9-tetrahydrocannabinol (105).
Tetracycline (127) and rifampin (126) have been shown to act synergisti
cally with amphotericin B to protect mice against N. fowleri infection. In
the tetracycline study, chemotherapy was started 72 hr after the mice had
 106 JOHN

been infected intranasally. Survival was 38% for amphotericin B-treated

mice and 88% for mice treated with the amphotericin B-tetracycline combi
nation (127). Combination therapy is a reasonable approach to treatment
for patients with PAM.

EPIDEMIOLOGY AND ECOLOGY

Geographic Distribution
Although it is a relatively rare disease, PAM has been reported worldwide.
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

Most of the reports have been from developed rather than from developing
nations, perhaps because of a greater awareness of the disease in these
countries and not because of greater incidence.
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Cases have been reported from Belgium, Czechoslovakia, Great Britain,

Northern Ireland, India, Australia, New Zealand, New Guinea, Uganda,
Nigeria, Venezuela, Panama, Puerto Rico, and the United States. As of
October 1981, 108 cases of Naeg/eria infection have been described world
wide; 49 of these have been from the United States. The states reporting
cases have been New York, Virginia, North Carolina, South Carolina,
Georgia, Florida, Mississippi, Arkansas, Texas, Arizona, Nevada, and Cali
fornia.
The majority of patients with naeglerial infection have had a history of
recent swimming in fresh water during hot summer weather. In Richmond,
Virginia, infection in 14 of the 16 cases probably was acquired in two
man-made lakes located within a few miles of each other (12, 51, 108).
Over a 3-year period, 16 young people died in Czechoslovakia after
swimming in the same heated, chlorinated, indoor swimming pool (23).
Similar fatal cases have been reported following swimming in swimming
pools in Belgium (68), England (11), and New Zealand (34); in hot springs
in California (15) and New Zealand (31); in lakes in Florida (10, 16), Texas
(16), Arkansas (143), and South Carolina (39); and in streams in Belgium
(129), New Zealand (32), and Mississippi (87).
Infection has not always been acquired through swimming, though. The
South Australia and northern Nigeria cases have occurred in rather arid
regions where swimming is not often indulged. The proposed means of
infection for these areas has been face washing and bath-related activity (2,
85) and inhalation of dust-borne cysts (84).

Environmental Isolations
Naegleriafowleri has been isolated from a variety of environmental sources.
During the summer of 1971, Nelson (98) isolated N. fowleri from a water
sample taken from a pond where a victim of PAM swam in 1967. This
constituted the first isolation of N. fowleri from an environmental source.
Today, the pond no longer is used for swimming but has since been con-
 NAEGLERIA MENINGOENCEPHALITIS 107

verted into an ornamental pool in a suburban housing development. How

ever, another man-made lake in the immediate neighborhood still attracts
swimmers daily throughout the summer months despite the fact that five
children have died of PAM after swimming there; the last case occurred 14
years ago in 1968 (12, 51, 108).
Additional environmental isolations of N. fowleri have been made from
tap wate:r in South Australia (69); heated indoor swimming pools in
Czechoslovakia (79) and England (11); lakes in Florida (139), Nigeria (86),
and Poland (80); warm effiuents from power plants in Florida (121), Texas
(121), and France (47) and from factories in Belgium (41); sewage and
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sludge samples in India (115), Korea (117), and Nigeria (86); soil samples
in South Australia (3), New Zealand (34), and Nigeria (86); and river water
in Spain (95).
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N. fowleri clearly is cosmopolitan in its environmental distribution. Curi

ously, though, most of the isolations have been made from habitats manipu
lated or altered in some way by man or, and this appears to be an important
factor, from habitats subjected to warming, whether man-made or natural.
On two occasions, N. fowleri has been isolated from the nasal passages
of young c;hildren. Amebae pathogenic for mice were isolated by nasal swab
from a 7-year-old boy in Richmond, Virginia (28, 112), and from two young
children, under 10 years of age, in Zaria, Nigeria (84). Although the Ni
gerian children had symptoms of upper respiratory tract infection, none of
the children had signs or symptoms of meningitis, and the Richmond child
had no evidence of infection of any sort.

Animal Infection
There are only a few reports, apart from those describing human infections,
in which Naegleria has been isolated from animals in nature. They include
the recovery of Naegleria from the gills of fish (122), from snails (81, 103),
and from various reptiles (57). Acanthamoeba, a related freshwater ameba,
has been detected in visceral lesions of a dog (4), in pulmonary lesions of
a buffalo {53), in a bull (96), in renal granulomas in a gold fish (131), and
cultured from clinical specimens and tissues of a variety of diseased domes
tic animals (77).
There is no evidence for the existence of an animal reservoir or carrier
host for N. fowled. Nonetheless, until adequate surveys have been carried
out, the possibility cannot be excluded. Animals that need to be examined
include aquatic insects (and insect larvae), crustaceans and mollusks, fishes,
amphibians and aquatic reptiles, waterfowl and shore birds, and aquatic or
diving mammals such as beavers, otters, and muskrats. All the above ani
mals could encounter N. fowler; naturally and, particularly the migratory
species, could serve as transport hosts. Perhaps this accounts for the obser
vation made by DeJonckheere & Van De Voorde (45) that N. fowleri could
 108 JOHN

be isolated from waters where old factories discharged thermal waste, but
not from the discharges of newer factories. The animal(s) involved may
require a period of adjustment before investigating a newly built facility.
Researchers have used several laboratory animal species as models for
their investigations of experimental PAM. Naegleria fowleri produces fatal
meningoencephalitis in mice (13, 18, 29, 92, and others), guinea pigs (18,
30, 104), rabbits (29), monkeys (29, .141), and sheep (144). The recent
significant finding that sheep are susceptible to N. fowled following intrana
sal instillation of amebae suggests the possible occurrance of PAM among
domestic livestock.
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Chick embryos also are susceptible to N. fowler; infection (66). Amebae

inoculated on the chorioallantoic membrane disseminate to the brain, liver,
spleen, and lungs. Inoculation with as few as 10 amebae will cause death
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of the chick embryo within 5 days.

The mouse is uniquely appropriate as an experimental model for studying
PAM. The basic features of the disease in man and mouse are the same with
respect to portal of entry, incubation period, and invasion of the nasal
mucosa, cribiform plate, and olfactory bulbs with subsequent spread to
more distant areas of the brain (92)
Fatal naeglerial meningoencephalitis in mice is route and dose dependent.
Most of the animal studies have employed either intranasal or intracerebral
routes of inoculation, which require fewer amebae to establish infection
than do the other routes. Clinical symptoms and death were produced in
mice inocluated intravenously and intraperitoneally with large doses of
amebae (1, 13). A dose of 5 X 106 amebae per mouse was administered
subcutaneously without deaths occurring (1). However, since the outcome
of an infection with N. fowleri is dose dependent, it is reasonable to propose
that a larger dose (or a more virulent strain) could produce the disease in
mice via subcutaneous inoculation.
It is also possible to produce infection in mice after intranasal instillation
with the flagellate stage of N. fowled (116). However, since flagellates are
rather unstable, they undoubtedly reverted to the ameba stage in the nasal
passages, and the amebae subsequently invaded the nasal mucosa. Flagel
lates were not seen in brain smears of mice dying from meningoencephalitis;
only amebae were noted (116).
Cysts of N. fowler; never have been observed in human brain tissue or
in tissues from experimentally produced animal infections. This is in direct
contrast to infection with Acanthamoeba in which cysts are routinely de
tected.

Control and Prevention

Because of the swimming-related nature of naeglerial infection, many swim
ming areas have been subjected to intense investigation. And although it is
 NAEGLERIA MENINGOENCEPHALITIS 109

true that N. fowleri has been isolated from some swimming areas (described
above), there are several reports in which environmental sampling of swim
ming pools has failed to produce N. fowleri (19, 42, 48, 70, 88). Obviously,
some factors favor the development of N. fowleri in swimming areas. These
include warm temperature, presence of an adequate food source (organic
matter or bacteria), insufficient residual free chlorine, minimal competition
from other protozoans, and probably optimal pH and oxygen levels.
With the present limited understanding of the ecology of N. fowleri,
practical measures for prevention and control of naeglerial infection include
education of the public, awareness within the medical community, and
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adequate chlorination (10 ppm) of public swimming facilities (44). Chang

(26) has demonstrated that N. fowleri amebae are sensitive to drying, high
temperature (>S1C), low temperature 1OC), and especially freezing.
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Cysts are sensitive to desiccation (nonviable in <5 min), survive poorly at

DOC, but tolerate high temperatures (Sl-6S0C). Cysts of N. fowleri have
been stored for up to 8 months with excysting amebae retaining virulence
(132). Effe:ctiveness of chlorination is dependent upon residual free chlorine,
temperature, and pH (26).
Legione/la pneumophila, the agent of Legionnaires' disease, has been
shown to be pathogenic for N. gruberi and N. jadini (107). It has been
suggested that Naegleria (and Acanthamoeba ) possibly may be natural
hosts for L. pneumophila, and that human infection may be acquired not
by inhaling free legionellae but by inhaling amebae full of legionellae (SO--
1000 or more bacteria per ameba) (107). Our own studies (D. T. John, N.
C. Mobley, unpublished data) indicate that N. fowleri is more susceptible
to infection by L. pneumophi/a than is N. gruberi. Perhaps Legione//a
(and/or other bacteria or viruses) serves as a natural biological control of
N. fowleri and accounts for the relative scarcity of N. fowleri in the environ
ment.
Conside:ring the millions of persons who swim each summer in streams,
lakes, and swimming pools, the probability of becoming infected is rather
remote. For example, despite the extensive distribution of N. fowleri in
Florida's freshwater lakes (121, 139), estimates are that the risk of acquiring
naeglerial infection'is about one case per 2.6 million exposures (138).

PHYSIOLOGY/METABOLISM

Nutrition and Growth

Perhaps the simplest way to grow N. fowleri is on the surface of non
nutrient agar (1.5%) spread with living or dead Enterobacter aerogenes or
Escherichi,a coli. Under these conditions, the amebae feed upon the bacteria,
and as growth enters stationary phase and the food supply is used up, they
 110 JOHN

begin to encyst. Cysts, if kept from drying out, will remain viable for
months, possibly years.
Unfortunately, the presence of bacteria too often hampers a variety of
quantitative studies. Therefore, several liquid media have been developed
for the axenic cultivation of N. fowleri (5, 17, 26 , 99). In general, these
media contain a phosphate buffer and either liver extract, yeast extract,
peptone, or casein derivative bases supplemented with serum.
Liquid axenic media developed by Balamuth (5), Cerva (17), Chang (26),
and Nelson & Jones (99) have been used for the cultivation of N. fowleri
(20, 40, 52, 133). Growth of N. fowleri under agitated and unagitated
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culture conditions was compared using these four axenic media (63). The
less enriched media of Cerva (17) and Nelson & lones (99) supported
greater cell yields under both agitated and unagitated culture conditions.
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The liver infusion-proteose peptone-yeast extract-glucose-calf serum me

dium of Balamuth (5) originally was developed for the axenic cultivation
of N. gruberi; hence, it is not surprising that it does not support good
growth of N. fowled. The two species are distinct organisms with different
nutritional requirements. In contrast to N. gruberi, N. fowleri grows best
in less enriched media (63, 110). Cerva medium contains only casitone and
horse serum (17) and Nelson medium has liver digest, glucose, and calf
serum (133).
By using agitated cultures and Nelson medium, it is possible to obtain
large quantities (3 x 109 amebae/liter) of N. fowled (133). At 37C, the
mean generation time is 5.5 hr for exponentially growing cells. There is only
slight utilization of glucose, and amino acids appear to serve as carbon and
energy sources.
Physical factors shown to affect the growth of N. fowleri in liquid axenic
cultures include pH, temperature, viscosity, and dissolved inorganic salts.
The pH optimum for growth initiation in agitated cultures is 5.5 (133), and
6.5 in unagitated cultures (21, 133). The pH of Nelson culture medium
increases about 2 U during 96 hr of growth (133). However, if the medium
is adjusted to maintain a constant pH, there is no change in growth of N.
fowleri, indicating that the pH increase of the culture medium does not limit
ameba growth (83).
Optimal temperature for the growth of N fowleri is dependent upon the
composition and concentration of the culture medium, viability of the
amebae in the inoculum, and size of the inoculum (20). It has been sug
gested that a threshold inoculum is necessary for growth initiation of N.
fowleri in broth cultures (40). The suggestion was made to account for
inconsistent ameba growth in cultures inoculated with less than 1()4 ame
bae/ml. However, the results of a study in which Basks are inoculated with
1()2-106 ameba/ml suggest that a threshold inoculum level does not exist
(83). Rather, there was a maximum population density or carrying capacity
 NAEGLERIA MENINGOENCEPHALITIS 111

for the medium. Cultures inoculated with 1()2 grew about 10 generations
before entering stationary growth phase, whereas cultures inoculated with
106 amebae/ml grew only one generation.
A 0.5% concentration of methylcellulose in liquid medium does not
inhibit th(: growth of N. fowleri (21) . By comparison, N. gruberi growth is
inhibited by concentrations of methylcellulose greater than 0. 2%.
Serum appears to be an important component of the liquid media used
for axenic: cultivation of N. fowleri. Various kinds and concentrations of
serum have been used. Several forms of calf serum and sera from other
vertebrate species have been evaluated for their ability to support growth
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of N. fowJ'eri in Nelson medium (64, 73). Of the 17 sera tested, calf serum
supported the greatest cell yields (1. 48 X 106 amebae/ml), whereas fetal calf
serum produced the lowest cell yields (2. 09 X lOs amebae/mI). Between
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these two and ranked in order of decreasing cell yield are pig, dialyzed calf,
monkey, newborn calf, lamb, turtle, dog, chicken, mouse, rabbit, frog,
horse, gamma globulin-free calf, fish, and human sera.
Hemin (1 ILg/ml) has been shown to replace serum as a growth require
ment for N. gruberi in axenic liquid cultures (6). However, the same is not
true for cultures of N. fowleri, although hemin, in addition to the serum,
will enhance growth (75). A semi-defined
taining he:min has been described (37) . A curious observation about the
Richmond, Virginia, cases is that 14 of the 16 infections were acquired in
two man-made lakes, which occur in the vicinity of the first iron smelter
to be built in the United States. The water in some of the small streams in
the area often appears reddish because of its iron content. Perhaps the
presence of iron or other heavy metals in nature provides an environment
favorable to the growth of N. fowleri.

Cell Diflerentiation

Naegleriafowleri is an ameboflagellate and, therefore, it is able to transform

from ameba to flagellate and revert to ameba, and to encyst and under
favorable conditions to excyst (Figure 1) . Yet, apart from testing to verify
the identity of N. fowleri isolates, there are no definitive physiological
studies of differentiation in N. fowleri. Cell differentiation in N. gruberi has
been reviewed by Fulton (58).
Trophozoites of N. fowleri are long and slender (8-15 ILm) and have
progressive flowing movement. The resting nucleus is spherical with a
sharply defined
Nuclear division is promitotic, in which the nucleolus elongates and divides
into two polar masses and the nuclear membrane remains intact (101) .
Cysts are spherical, often clumped closely together, and 7-10 ILm in
diameter (13). Ultrastructure examination reveals an average of less than
 112 JOHN

2 mucoid-plugged pores per cyst and a relatively thin cyst wall, a feature
that makes N. fowled cysts susceptible to desiccation (109).
When trophozoites are suspended in distilled water or non-nutrient buffer
(58) they transform into temporary flagellated forms. The flagellate has an
elongate, pear-shaped body, usually two flagella of equal length, a nucleus
in the narrower anterior region, and no cytostome (13, 101). The ultrastruc
ture of N. fowler; flagellates is that of a typical eucaryotic protist (102).
There is a distinct nuclear membrane and prominent nucleolus, numerous
vacuoles and cytoplasmic inclusions, pleomorphic mitochondria, and some
rough endoplasmic reticulum. Amebae of N. fowler; (LEE strain) became
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

flagellates 150 to 180 min after transfer to non-nutrient buffer. Basal bodies,
a rootlet, and flagella are formed quickly after an initial lag of 90 min.
Carter (13) observed that transformation occurred after 20 hr in distilled
by University of Tasmania on 09/03/14. For personal use only.

water, and that the flagellates persisted until 48 hr. Transformation was
unreliable below 21C and was better, with 50% flagellates, at 37C.
We have noted (D. T. John, N. C. Mobley, unpublished data) that several
of our axenically cultured N. fowleri isolates no longer differentiate into
flagellates when amebae are placed in non-nutrient buffer (Table 1). Appar-
Table 1 Differentiation of Naegieria fowleri amebae to flagellatesa

Isolation
Flagellates Maximum
t ran sfo r mat ion
b
Strain of ameba Location Da t e Ref. (%)
------

Lov ell Florida 1974 40 65 5

KUL Belgium 1973 129 53 4
LEE(M-Il)c Virginia 1968 51 52 4
LEE(ATCC-30894) Virginia 1968 51 31 4
TY Virginia 1969 51 22 5
6088 California 1978 15 20 5
WM Virginia 1969 51 17 4
HB-5 Texas 1977 None 14 5
CJ Virginia 1967 51 12 5
0 359 Belgium 1970 68 2 4
GJ Florida 1972 139 <I 4
HB-4 North Ca rolin a 1977 None 0
HB- 4(M-8)c North Carolina 1977 None 0
NF66 A u stra li a 1966 13 0
NF69 Australia 1969 13 0
aAmebae were grown at 37C in Nelson medium with 2% calf serum. At 72 hr, medium
was removed, amebae were rinsed three times with Page saline, covered with cold (4C)
Page saline, and vortexed. Amebae ( 106 cells/ml) were placed in 125-ml Erlenmeyer
flasks and shaken at 37C in a gyrotory shaker (New Brunswick) at 100 rpm. Flasks were
examined hourly for 8 hr by placing samples in f)'Antoni', iodine and counting the per
centage of flagellates. Cultures not producing flagellates were held and examined period
ically to 36 hr.
bTime in hours after the initiation of differentiation (time zero ).
cM11 and M-8 denote amebae that had been serially mouse-passaged II and 8 times,
respectively.
 NAEGLERIA MENINGOENCEPHALITIS 113

ently, some axenic populations of N. gruberi amebae fail to transform (100)

and must be maintained with bacteria to do so. Fulton (58), however, states
that axenil()ally grown N. gruberi differentiate to flagellates but do so more
slowly than bacteria-grown cells. Whatever the reason, it would be instruc
tive to examine transformation in our non-differentiating N. fowleri isolates
after growth with bacteria.

Macromolecular Composition
Changes in cell composition of N. fowleri are related to culture age (135).
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

For agitated axenic cultures, average cell dry mass remained constant dur
ing log growth at 150 pglameba, but decreased 30% during stationary
growth at 96 hr. During log growth 80-85% of the cell dry mass was
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protein (120 pg/ameba). Cell dry mass and protein of N. fowleri are about
70% of vaJues reported for N. gruberi (134).
During log and stationary growth phases, carbohydrate content averaged
15 pglamt:ba, and RNA was about 18 pglameba (135). RNA content for
N. gruberi was about 8 pglameba (134). Perhaps the over twofold greater
RNA value for the smaller pathogenic N. fowleri reflects different biosyn
thetic capabilities by maintenance of a larger ribosome complement.
Total DNA content was 0.2 pg/ameba during log growth; it doubled
during transition from log phase to stationary phase and then gradually
decreased to nearly initial levels. The peak in DNA content corresponded
to an incre:ase in the average number of nuclei per ameba; nuclear number
then decreased as cells entered stationary growth phase (135).

Cell Membrane and Agglutination

Concanavalin A (Con A) agglutinates N. gruberi but does not agglutinate
N. fowleri (76, 120). Agglutination is time and temperature dependent and
Con A concentration and ameba concentration dependent over certain
ranges. At least 106 amebae/ml are needed for maximum agglutination, and
Con A concentrations higher than 100 JLg/ml do not appreciably increase
agglutination. These results indicate that only N. gruberi has appreciable
quantities .of N-acetyl glucosamine and mannose-like residues manifested
on the cell surface. Naegleria lovaniensis, which is nonpathogenic for mice,
is also agglutinated by Con A (118).

Respiratory Metabolism
As an oPPClrtunistic parasite, N. fowleri lives in an oxygen-rich environment
(the brain) and so one would expect it to have an aerobic metabolism. The
synonym N. aerobia (114) recognized the aerobic nature of the organism,
in contrast to the anaerobic nature of strictly parasitic amebae. Unlike
Entamoeba histolytica, an anaerobic parasitic ameba that lacks mito-
 114 JOHN

chondria (59), N. jowleri lives in aerobic aqueous environments and has

many mitochondria (90, 111).
Whole cell respiration rates were measured polarographically throughout
the growth cycle of N. jowleri (136). Under agitated culture conditions,
amebae consumed 30 ng of O/min/mg of cell protein during log growth.
Under similar conditions N. gruberi amebae consumed 80 ng orO/min/mg
of cell protein (137). The lower oxygen consumption, and most likely
oxygen requirement, by N. jowleri probably explains the presence of the
pathogen in heated waters where dissolved oxygen concentrations are sub
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

stantially reduced.
Respiratory rate gradually declined during stationary growth phase. The
reduction in respiratory rate may involve respiratory control since further
by University of Tasmania on 09/03/14. For personal use only.

increases in respiratory rate did not occur in spite of fresh oxygen supplies
(136).
The respiratory process of isolated N. jowleri mitochondria is similar to
classical mammalian cell mitochondria. Oxidation was "coupled" to phos
phorylation (ATP formation) as shown by the two- to threefold increase in
respiration upon addition of phosphate acceptor or uncoupling agent.
Difference spectra of oxidized and dithionite-reduced mitochondria showed
distinct absorption bands of ftavins, c-type. b-type. and a-type cytochromes
(136).

VIRULENCE AND IMMUNITY

Mechanisms of Pathogenesis
The determinants of virulence, invasiveness, and pathogenicity of Naegleria
jowleri are largely unknown. Electron microscope studies of experimentally
induced PAM in mice have demonstrated phagocytic activity by amebae
(90, 130).
N. jowleri causes a destructive cytopathic effect in cultured mammalian
cells, and it is generally supposed that this cytopathic activity is associated
with trophozoite pathogenicity. Opinion, though, is divided on the mecha
nism of cell damage. Chang (25) reported that supernatant medium from
N. jowleri-infected cultures of mammalian cells induced cell degeneration
when fresh cultures were inoculated with it, which suggests that amebae
secrete cytolytic or cytotoxic enzyme-like substances. He concluded (27)
that the pathogenicity of cytopathic effect of N. jowleri can be attributed
to a phospholipolytic enzyme released by the amebae during active growth.
Wong et al (142) observed differences in cytolytic enzyme synthesis be
tween highly virulent and low-virulent strains of N. fowleri; for example,
highly virulent strains produced a magnitude more catalase than low-viru
lent strains. Cursons et al (33) found that N. fowleri produced a greater
 NAEGLERIA MENINGOENCEPHALITIS 115

amount of phospholipase A than did nonpathogenic N. gruheri. Hysmith

et al (67} have measured sphingomyelinase levels in Naegleria culture
media and determined that sphingomyelinase activity was approximately
lOO-fold greater in culture medium of virulent N. fowl eri than it was in
culture media of low-virulent N. fowler; or N. gruberi. Increased levels of
sphingomyelinase activity in culture medium may be directly associated
with the demyelination in brain and spinal cord that has been described in
PAM (27, 51).
Brown (7, 8) suggests that the cytopathogenicity of N. fowler; in second
ary mous(:-embryo cells does not involve ameba-associated cytotoxic activ
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

ity, but depends on normal phagocytic function. Amebae that were

immobilized and agglutinated by specific antiserum exhibited no cytopathic
activity, allthough they remained alive and were in constant contact with the
by University of Tasmania on 09/03/14. For personal use only.

host cells. Cytochalasin B, shown to inhibit phagocytosis in amebae, inhib

ited the cytopathogenicity of N. fowleri when it was added to cell culture
medium. Brown concludes that N. fowleri amebae attack and destroy cul
tured mouse-embryo cells by a phagocytosis-like mechanism alone, without
the aid of ameba-associated cytotoxic or cytolytic agents.
We have examined the susceptibility of various mammalian cell lines to
the cytopathic activity of a highly virulent strain (HB-4) of N. fowleri. With
a multiplicity of infection of 1, complete destruction of the monolayers
occurred from 24 hr (BHK-21, Vero, WI-38 cells) to 72 hr (L929,
Ktk-, Neuro-2a cells) after inoculation of amebae for cultures incubated
at 37C (Table 2). Cytopathic effect occurs to a lesser extent and later for
cultures inoculated with a low-virulent strain of N. fowleri.
Brown (9) also has described the cytopathogenicity of nonpathogenic
N. gruheri in several mammalian cell lines, cultured at 30 rather than
37C. Again, cytopathic effect was caused by phagocytosis, and he sug
gests that temperature sensitivity has been a significant factor in the re
ported differences in cytopathogenicity between N. fowler; and N. gru
heri amebae.
Naegleria-induced cytopathic effect has also been attributed to the trans
mission of infectious cytopathogenic material from ameba to susceptible
avian and mammalian cultured cells (52). The infectious material, present
in both N. fowler; and N. gruberi, appears to be a protein with an estimated
molecular weight of 50,000. It is capable of sustaining itself in cell culture
and in serial passage through multiple dilutions.
Several factors have been shown to affect the virulence of N. fowler; for
mice; they include incubation temperature, growth phase, and strain of
ameba (61). Amebae cultured at 30 and 37C were more virulent than
amebae growth at 23 and 44C. Mortality was greater for mice inoculated
with amebae harvested at late logarithmic and early stationary growth
 116 JOHN

Table 2 Naegleria Jowleri (HB-4 strain)-induced cytopathic effect (CPE)

in cultured mammalian cellsa

Time (hr) for

maximum ePEe

Celliineb Animal species and tissue 24 48 72

BHK-2l Baby golden hamster kidney IV

Vera African green monk ey kidney IV
WI-38 Human lung IV
CF-3 Human foreskin IV
IV
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

CHO-Kl Chinese hamster ovary

HeLa S3 Human epithelioid carcinoma, IV
cervix
HEp-2 Human epidermoid carcin oma , IV
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larynx
Mv 1 Lu Mink lung IV
NB41A3 Mouse neuroblastoma IV
L929 Mouse connective tissue IV
Ltk- M ouse connec tive ti ssu e IV
Neuro-2a Mouse neurob l as t oma IV
aHB-4 is a highly virulent strain of N. fowleri isolated by R. B. Finley in
July 1977 from patient sp inal fluid, before death, at Winston-Salem. NC.
b Cells were grown and maintained in MEM with fetal calf serum. All c ell
lines were obtained from the American Type Culture Colle ction (Rockville,
MD) except CF-3 (Noble Fdn., Ardmore, OK) and Ltk- (S. Kit, Baylor,
Houston, TX), a th ymi dine kinase d efi cie o t L929 derived cell line.
CM ulti plicit y of infection was 1; four 25-cm2 tissue culture flasks per
cell line; 37C incubation temperature; ePE of IV represents complete
breakdown of monolayer so that culture flask contains only am eb ae and
cellular debris.

phases than it was for amebae harvested at early logarithmic and late
stationary growth phases.
The incubation of N fowled amebae with mouse anti-N fowleri serum
has been shown to have a rather dramatic effect upon ameba virulence and
subsequent mouse mortality after intravenous inoculation (62). The mortal
ity rate was 15% for mice inoculated with immune serum-treated amebae
compared with 95% mortality rate for mice inoculated with amebae in
cubated in normal (nonimmune) mouse serum. Viability testing, using try
pan blue exclusion, immediately after incubation of amebae with serum,
showed that the immune mouse serum caused greater damage to the
amebae, with -5% nonviable cells as compared to less than 1 % nonviable
amebae after incubation with normal mouse serum.
Virulence varies greatly among the different human isolates of N. fowleri.
Table 3 gives the mortality for mice inoculated intranasally with 5 X 103
amebae/mouse of 13 different strains (isolates) of N. fowleri. Cumulative
percent mortality at 28 days ranged from 0-100% and mean time to death
 NAEGLERIA MENINGOENCEPHALITIS 117

Table 3 Mortality for mice inoculated intranasally with 5 X 103 amebae/mouse of 1 3

strains of Naegleria fowleria

Initial Date of Cumulative Mean lime

Strain of ameba isolation isolation Ref. percent dead b to death (hr)

CJ Virginia 1 967 51 Oc
KUL Belgium 1973 1 29 Oc
TY Virginia 1 969 51 10 14.0
0 359 Belgium 1 970 68 15 1 3.3
LEE(A TCC-30894) Virginia 1 968 51 20 1 7.8
NF66 Aus tral ia 1 966 13 75 1 6.8
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

Lovell Florida 1 974 40 85 15.5

HB-4 North Carolina 1977 None 85 7.2
WM Vir gin ia 1969 51 90 1 1.1
NF69 Australia 1969 13 90 10.6
by University of Tasmania on 09/03/14. For personal use only.

GJ Florida 1972 139 90 9.4

6088 California 1 978 15 90 9.6
HB-5 Texas 1977 None 95 8.1
d
LEE(M-IO) Virginia 1 968 51 1 00 1 4.0

aThcrc wcrc 20 male DUB/IeR mice per group (13-18 g).

b Recorded to 28 days after inoculation.
CMortality occurred with increased dosage.
dThe M-IO denotes LEE strain amebae that had been serially mousepassaged 10 times.

for the 13 strains was from 7.2 to 17.8 days. Kadlec (78) describes the great
variation that occurred in virulence for 33 strains of N. fowleri isolated from
the water of an indoor swimming pool. Naegleria fowleri amebae have been
shown to differ in their susceptibility to trimethoprim, and the difference
appears to be related to ameba virulence (22). The growth of virulent strains
was unaffected by trimethoprim (400 f'glml of medium), whereas avirulent
isolates were completely inhibited by the drug at concentrations of 4 f'glml.
Trimethoprim susceptibility may be a useful aid in the selection of virulent
environmental isolates of N. fowleri.
Ameba virulence appears to be related to the length of time a strain has
been maintained in axenic culture (61). Axenic cultivation gradually de
creases the virulence of N. fowleri. However, virulence can be enhanced and
perhaps restored to original levels by passage of amebae in mammalian cell
culture and by serial mouse passage (43, 142).

Resistance and Susceptibility

The factors responsible for innate resistance or susceptibility to naeglerial
infection are undefined. Relatively few human infections have occurred
even though large numbers of individuals have been exposed to similar
environmental conditions. Most cases of PAM have occurred in previously
healthy children or young adults.
 118 JOHN

When examining naeglerial infection in mice, it is readily apparent that

the age of the test animals dramatically affects the outcome of N. fowleri
exposure. Young mice are uniformly susceptible, but as they become older
they also become increasingly more resistant to infection. Female mice have
been shown to be more resistant to naeglerial infection than male mice of
the same age and strain (60). Interestingly, there have been more male than
female victims of PAM. This generally has been attributed to the vigorous
swimming and diving and adventuresomeness of males and, thus, the proba
bility of greater exposure to N. fowleri rather than to the possibility of
greater susceptibility. Perhaps males, indeed, are more susceptible to infec
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

tion, and specific hormones (or hormone levels) are involved. To date this
area of research has not been investigated.
Different mouse strains vary greatly in their susceptibility to infection
by University of Tasmania on 09/03/14. For personal use only.

with N. fowled (43, 60). In both these studies, Cs7BI mice were the most
resistant of the strains tested. One especially susceptible mouse strain (A/
HeCr) is deficient in the C'5 component of complement (60). Obviously, it
is premature to suggest that complement is involved in host resistance.
Nonetheless, N. fowleri has been shown to be an efficient activator of serum
complement by either the classical or alternative pathways (65, 106).
Serum obtained before death from a victim of PAM was assayed for
immunoglobulin (Ig) levels (38). Although IgG and IgM levels were within
normal limits, the IgA value was low. However, a second report from
England (11) revealed normal levels of IgA for serum taken from a patient
on day 3 of a fatal infection. In both patients, serum was measured, not
secretory IgA levels. Because serum IgA levels may not be an accurate
reflection of secretory IgA concentrations, both patients may have had
deficient levels of secretory IgA. Serum IgA levels are greatly increased in
mice given three intranasal inoculations of living N. fowleri (62, 72).

Immunization and Protection

Mice have been immunized with living, formalinized, and freeze-thawed
N. fowleri via subcutaneous, intraperitoneal, intravenous, and intranasal
routes of inoculation (74, 128). Protection of mice against lethal intranasal
or intravenous challenge ranged from 6 to 55%. Intravenous immunization
with N. gruberi afforded 65% protection (74), and three intranasal immu
nizing doses with N. gruberi produced 88% protection against intranasal
challenge with N. fowleri (71). Nonpathogenic N. gruberi appears to be a
better immunogen than N. fowleri. Perhaps man's unwitting exposure to
N. gruberi, abundant in freshwater environments, affords protection against
what normally would be a lethal exposure to N. fowleri.
Modest protection has been achieved by intraperitoneal immunization of
mice with N. fowleri culture supernatant (124). Controls, receiving equal
 NAEGLERIA MENINGOENCEPHALITIS 119

volumes of fresh culture filtrate, did not exhibit protection. Presumably,

protection was afforded by antigenic material derived from the amebae
during cultivation. To date, all immunization attempts have produced only
incomplete protection; solid immunity has not yet been achieved.
The mechanisms for protective immunity only recently have been given
consideration. Protection against naeglerial infection can be transferred in
mice by immune serum but not by sensitized spleen cells (1 25 ). Antibody
to N. fowleri has been detected in surveys of normal human sera. Using a
radioimmunoassay, the response against intracellular antigens was higher
than the response against cell surface antigen (1 23). The indirect fluores
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

cent-antibody test demonstrated titers ranging from I : 5 to I : 20 for 9 3

serum samples tested (35 ).
The role of cell-mediated immunity has been partly examined. A delayed
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hypersensitivity reaction has been described for guinea pigs sensitized by the
soluble antigens of freeze-thawed N. fowleri (4 6 ) and by freeze-thawed
amebae plus complete Freund adjuvant (142). Macrophage inhibition by
the lymphokine macrophage inhibition factor has been described for N.
fowleri (36).
Naegleria fowleri amebae have been shown by immunofluorescence to
cap and nemove or internalize surface-bound antibody (5 4). The ability of
N. fowleri to remove antibody from the cell surface may enable the amebae
to counter the host's immune defenses.
Neutrophils from N. fowleri-immunized mice are capable of killing
amebae (55 ). One method of killing is for a group of neutrophils to surround
an ameba and destroy it, presumably by contact and release of enzymes onto
the ameba cell membrane. However, a novel phagocytic process has been
described in which neutrophils pinch off portions of an ameba. Although
unable to phagocytose an entire ameba, several neutrophils are able to
rupture an ameba by pinching off and engulfing portions of it (55 ).

CONCLUDING REMARKS

Primary amebic meningoencephalitis, a fatal human disease caused by Nae

gleria fowleri, is known only as a somewhat rare or exotic disease, in short,
a medical curiosity. However, circumstantial evidence suggests that human
meddling with water resources may exaggerate the problem. If pollution
and/or treatment of water increases the population of N. fowleri in public
waters, then man and animals dependent upon impounded waters may be
at serious and growing risk.
The determinative factors in virulence and host resistance to naeglerial
infections are unclear in both human and experimentally induced PAM.
Relatively few human infections have occurred, even though large numbers
 120 JOHN

of individuals have been exposed to similar environmental conditions. And

most of these infections have occurred in previously healthy children or
young adults. It is not known whether the higher incidence of disease
among young males reflects behavioral attributes (adventurous, vigorous
swimming and diving) or physiological factors (hormone levels, immuno
logical competence).
The virulence factors that contribute to the pathogenesis of N. fowler; are
largely undefined. In vitro studies have implicated toxins and cytopathic
enzymes, infectious cytopathogenic material, and phagocytosis, a natural
function of all amebae. Perhaps it is not unreasonable to expect all of these
Annu. Rev. Microbiol. 1982.36:101-123. Downloaded from www.annualreviews.org

factors to be involved in pathogenesis within the infected host.

Two recent cases of fatal meningoencephalitis resulting from free-living
amebae have been described in which the organisms could not be identified
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positively as either Naegleria or Acanthamoeba (50, 94). The genus Vahl

kampjia has been suggested as an alternative. The significance of these two
cases is that given the right conditions, serious disease in man (and possibly
animals) may be produced by free-living amebae other than those we have
come to regard as pathogenic.

ACKNOWLEDGMENTS

I am pleased to dedicate this review to Dr. Cecil B. Hamann of Asbury

College who first introduced me to the subject of medical parasitology. My
research has been supported by grant 771019 from A. H. Robins Co.,
Richmond, Virginia, and by Public Health Service grant AI 18788 from the
National Institute of Allergy and Infectious Diseases.

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